Hepatitis C remains a serious health threat, with few therapeutic options. Amplification of the positive-stranded viral genome is regulated by highly conserved RNA elements located in the viral 5' and 3' noncoding regions. Translation of the viral proteins is mediated by an unusually divergent internal ribosome entry site (IRES) located in the viral 5' noncoding region. The IRES is a conserved 320 nucleotide RNA that binds directly to ribosomal 40S subunits to direct translation initiation. Initiation of viral minus-stranded RNAs is modulated by a conserved landscape of RNA stem-loop structures located at the very 3' end of the viral RNA genome. Here, a combined genetic, biochemical, biophysical and structural approach is proposed to determine the mechanism by which the viral noncoding regions control HCV translation and replication. In specific aim 1, biochemical and genetic methods will delineate the mechanisms of HCV translation initiation, and will unravel the roles of a cellular microRNA in modulating gene expression of the viral RNA. The mechanistic information, coupled with a large amount of preliminary spectroscopic data, will be used in specific aim 2 to guide NMR structure determinations of IRES domains and the full-length intact IRES. To complement static structural experiments, specific aim 3 explores the conformational dynamics of free and ribosome-bound IRES RNA using single-molecule fluorescence spectroscopy, and mechanistic details will be obtained by direct observation of tRNA binding and release during translation initiation. The role of the 3' noncoding in IRES-mediated function will be also monitored by single-molecule fluorescence to detect transient end-to-end communication in viral RNA. Similarly, the effects of microRNA-viral RNA interactions on conserved sequence elements located at the 3' end of the viral genome will be examined using single-molecule fluorescence. The results of this proposal will have direct impact on our understanding of critical steps in the viral replication cycle, and the possible development of novel antiviral therapies.